1. Field of the Invention
The invention relates to oligoside derivatives, their process of preparation, and their applications.
2. Description of the Related Art
Natural oligosides are able to be prepared in free form from various physiologic liquids such as milk, or extracts from natural or transformed products (honey, beer, etc.). Natural oligosides are also able to be obtained by cutting a glycoside bond from one of the sugar moieties of glycoconjugates (glycolipids, glycoproteins, polyosides, proteoglycans, etc.), by hydrolysis with the aid of enzymes or by chemical catalysis from said glycoconjugates.
The natural oligosides are able to be used as substrates, as inhibitors, as recognition signals, etc. In the majority of cases, it is advisable to fix the oligoside on a molecule, matrix or particle, which can be chosen from:
a matrix as a support for affinity chromatography; PA1 a bead of gold or latex, for histology and cytology; PA1 a protein for visualilzation, purification, etc., in particular 1) specific receptors of osides, receptors which are called lectins, adhesins, agglutinins, etc., or 2) proteins with or without enzymatic activity, which have an affinity for the osides, in particular the glycosyltransferases, exoglycosidases or endoglycosidases PA1 a lipid for the characterization of the preceding receptors; PA1 oligonucleotides for selectively increasing their capture by targeted cells; PA1 a protein or polymers for the targeting of drugs, oligonucleotides or genes, or for obtaining intramolecular inhibitors. PA1 a=0 or 1, PA1 j=0 or 1, PA1 b=0 or 1, PA1 p=2 to 4, in particular 2, PA1 provided that PA1 D represents a residue of an organic acid of the formula DCO.sub.2 H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in particular CH.sub.3, PA1 Z represents PA1 X represents: ##STR2## PA1 m being an integer from 0 to 10, preferably from 0 to 5 and advantageously 1 or 2, k=0 or 1 PA1 Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5, O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, ##STR3## PA1 R representing a group possessing an alcohol, phenol, thiol, or amine function, PA1 P being such as is defined hereafter, PA1 A.sub.i representing an organic radical such as an alkylene chain of 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n --W--(CH.sub.2).sub.n', n+n' representing an integer from 0 to 10, W representing CHY, Y being H, an alkyl from 1 to 6 linear or branched carbon atoms, an .alpha. amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenylgroup, PA1 P, P' and P" are identical or different and represent: PA1 P, P' and P" possessing at least one function allowing a condensation reaction by reaction with an oligopeptide, for example PA1 provided that if Z represents B'--P', and/or X comprises P and/or P" in the formula. PA1 to a molecule, matrix or particle, by the intermediary of either a functional group X, or a functional group Z, PA1 or to two molecules, matrices or particles, by the respective functional groups X and Z, or by the intermediary of two functional groups X, or to three molecules, matrices or particles, by the intermediary of functional groups X and Z. PA1 a single oligoside fixed to a molecule, matrice, or particle, P, PA1 a single oligoside fixed to a molecule, matrice, or particle, P', PA1 a single oligoside fixed to a molecule, matrice, or particle, P", PA1 a single oligoside fixed to two molecules, matrices, or particles, P and P' respectively, or P' and P", or P and P', PA1 a single oligoside fixed to three molecules, matrices, or particles, P, P', and P". PA1 a=0 or 1, PA1 j=0 or 1, PA1 b=0 or 1, PA1 p=2 to 4, in particular 2, PA1 provided that PA1 D represents a residue of an organic acid of the formula DCO.sub.2 H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in particular CH.sub.3, PA1 B represents H, an alkyl of 1 to 10 carbon atoms, or a side chain of an .alpha. amino acid such as CH(CH.sub.3).sub.2, CH.sub.2 OH, CH.sub.3, and preferably H, PA1 X represents: PA1 m being an integer from 0 to 10, preferably from 0 to 5 and advantageously 1 or 2, R and P being as defined hereinafter, PA1 A.sub.i represents an organic radical such as an alkylene chain of 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n --W--(CH.sub.2).sub.n ', n+n' representing an integer from 0 to 10, W representing CHY, Y being H, an alkyl from 1 to 6 linear or branched carbon atoms, an .alpha. amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenyl, PA1 R represents a group possessing an alcohol, phenol, thiol, or amine function, PA1 P represents: PA1 A.sub.i, m, R, P and P" having the significations indicated above. PA1 a=0 or 1, PA1 j=0 or 1, PA1 b=0 or 1, PA1 p=2 to 4, in particular 2, PA1 provided that PA1 D represents a residue of an organic acid of the formula DCO.sub.2 H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in particular CH.sub.3, PA1 Z.sub.1 represents PA1 X.sub.1 represents PA1 R representing a group possessing an alcohol, phenol, thiol, or amine function, PA1 Q representing OH, OCH.sub.3, OCH.sub.2 --C.sub.6 H.sub.5, O--C.sub.6 H.sub.5, O--C.sub.6 F.sub.5, ##STR15## PA1 m being an integer from 0 to 10, preferably from 0 to 5 and advantageously 1 or 2, PA1 A.sub.1 represents an organic radical such as an alkylene chain from 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n --W--(CH.sub.2).sub.n', n+n' representing an integer from 1 to 10, W representing CHY, Y being H, an alkyl from 1 to 6 linear or branched carbon atoms, an .alpha. amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenyl. PA1 a=0 or 1, PA1 j=0 or 1, PA1 b=0 or 1, PA1 p=2 to 4, in particular 2, PA1 provided that PA1 D represents a residue of an organic acid of the formula DCO.sub.2 H, in particular H or an alkyl chain of 1 to 10 carbon atoms, in particular CH.sub.3, PA1 B represents H, an alkyl chain of 1 to 10 carbon atoms, or a side chain of an .alpha. amino acid such as CH(CH.sub.3).sub.2, CH.sub.2 OH, CH.sub.3, and preferably H, PA1 m being an integer from 1 to 10, preferably from 0 to 5 and advantageously 1 or 2, PA1 A.sub.1 represents an organic radical such as an alkylene chain from 1 to 10 carbon atoms, in particular (CH.sub.2).sub.n --W--(CH.sub.2).sub.n', n+n' representing an integer from 1 to 10, W representing CHY, Y being H, an alkyl from 1 to 6 linear or ramified carbon atoms, an .alpha. amino acid residue, natural or synthetic, or W representing an aromatic compound, in particular phenyl, PA1 R represents a compound possessing an alcohol, phenol, thiol or amine function. PA1 --NH-pC.sub.6 H.sub.4 --N.dbd.C.dbd.S and its precursors: PA1 R can also represent ##STR23## PA1 --NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--CH.sub.2 -T PA1 T=Br, I, Cl PA1 --NH--CH.sub.2 --CH.sub.2 --NH--CO--CH.sub.2 --(CH.sub.2).sub.m --S--S-Pyr PA1 N.B.: -Pyr: -2-pyridine PA1 --NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --S--S-Pyr PA1 R can also represent: ##STR26## PA1 R can also represent: ##STR27## PA1 R can also represent: PA1 --NH--(CH.sub.2).sub.m -pC.sub.6 H.sub.4 OH PA1 --NH--CH.sub.2 --(CH.sub.2).sub.m --CH.sub.2 --NH--CO--(CH.sub.2).sub.m -pC.sub.6 H.sub.4 OH PA1 R can also represent: ##STR32## PA1 simple or complex osides recognized by the lectin membranes, and chosen from: PA1 a. Asialo-oligoside of type lactosamine triantenna: receptor of asialoglycoprotein ##STR38## PA1 b. Asialo-oligoside of type lactosamine tetraantenna: receptor of asialoglycoprotein ##STR39## PA1 c. Lewis.sup.x : LECAM 2/3 ##STR40## PA1 d. Sialyl Lewis.sup.x : LECAM 3/2 ##STR41## PA1 e. Derivative of Lewis.sup.x sulfate (HNK1): LECAM 1 ##STR42## PA1 f. Oligomannoside: receptor of mannose ##STR43## PA1 g. Phosphorylated oligomannoside: receptor of mannose 6 phosphate ##STR44## PA1 h. Oligosaccharide of sulfated lactosamine type: receptor of sulfated GalNAc 4 ##STR45## PA1 one condenses an oligoside having a free terminal reducing sugar, on the nitrogen atom of an intermediary molecule, this nitrogen atom belonging to the amine group, linked to a carbon atom placed in .alpha. of a C.dbd.O group, the intermediary molecule possibly possessing a lateral chain containing a functional group such as OH, SH, NH.sub.2 or COOH, free or protected, this intermediary molecule being chosen from the following intermediary molecules: a amino acid, natural or synthetic, derivative of .alpha. amino acid, amino acid in N-terminal position of a peptide, or a peptidic derivative, possibly in presence of a catalyst such as imidazole, in a solvent appropriate for obtaining a derivative of glycosylamine in which the terminal ose of the oligoside conserves its cyclic structure, and in which the semiacetalic hydroxyl is replaced by the .alpha. amine of one of the said starting molecules, PA1 while the intermediary molecule does not possess a lateral chain containing a functional group such as described above, or a side chain in which the functional group is possibly protected, one acylates the derivative of glycosylamine obtained at the issue of the preceding step by the addition of an organic acid activated by a classical activator such as carbonyl diimidazole, BOP (benzotriazolyl N-oxy-tris(dimethylamino) phosphonium hexafluorophosphate) or HBTU (O-benzotriazol-1-yl-N,N,N',N',tetramethyluronium hexafluorophosphate) to obtain a derivative of N-acylated glycosylamine, followed possibly by a deprotection of the functional group of the said lateral chain, in view of a possible substitution, PA1 while the intermediary molecule possesses a side chain containing a carboxylic group, one activates the said carboxylic group to induce an intramolecularly reaction with the said .alpha. amine, leading to a cyclization inside the said intermediary molecule, to obtain a derivative of N-acylated glycosylamine. PA1 while the intermediary molecule possesses a side chain containing a carboxylic group, one can add the acylation agent in an active ester form. PA1 R.sub.1 representing a residue of an organic molecule without a protected functional group, or R.sub.1 being also able to represent H; PA1 R.sub.2 representing a residue of an organic molecule such as --CO--R2, either an ester or an amide; PA1 R.sub.3 representing a residue of an organic molecule preferably not comprising a free functional group. PA1 R.sub.3 representing a residue of an organic molecule comprising a second functional group such as, in particular SH, free or protected, or --CO.sub.2 H. PA1 R.sub.2 and R.sub.3 having the significations indicated above, and R.sub.4 representing a residue of an organic molecule possessing a functional group, specifically a carboxylic group, R.sub.4 representing specifically CH.sub.2 --CH.sub.2 --CO.sub.2 H. PA1 R.sub.2 having the significations indicated above, PA1 R.sub.5 --CO.sub.2 H being a residue of an organic molecule such as --(CH.sub.2).sub.n --CO.sub.2 H PA1 n being an integer from 1 to 10, preferably 2 or 3.
The synthesis of derivatives of oligosides able to be linked by covalent means to a protein, a matrix, an oligonucleotide, or by general means to an organic molecule or a particle, in all cases preserving the integrity of the structure and functions of each of its sugar components--which is necessary for preserving the functional capacity of the oligoside can be obtained essentially in two ways: the total synthesis de novo and the intermediary transformation into glycosylamine. A third way which leads to an equally useful derivative but which destroys the terminal reductorose is amination in a reducing medium.
Concerning total synthesis, this requires a selective protection of the hydroxyls non-engaged in a glycoside bond, steps of condensation, steps of selective deprotection, and a step of final deprotection. Even if over the last decades the yields of some of these steps were able to be improved, this is a long process and the overall yield remains modest, and this all the more as the oligoside to be synthesized is more complex.
The yields for each step are between 20 and 95% according to the steps considered.
For example, the synthesis of a para-nitrophenyl derivative of a pentasaccharide such as the determinant of Lewis x: EQU Gal.beta.3(Fuc.alpha.4)GlcNAc.beta.3Gal.beta.4Glc.beta.-O-p-C.sub.6 H.sub.4 --NO.sub.2
requires in total several tens of steps. Each step has a yield of between 50 and 95%, more generally 80%. In total the yield of the product sought is of some %.
The elevated number of steps arises from the fact that the alcohol functions of each ose must be protected in a different manner depending on whether the hydroxyl under consideration will or will not be implicated in a condensation reaction.
A sugar such as GlcNAc which will be substituted 2 times will receive momentarily 3 different substitutes in order to permit a selective substitution on the hydroxyl 3 by galactose, on the hydroxyl 4 by fucose, the hydroxyl 6 remaining protected until the final deprotection.
For each step of condensation, the yield is affected by the fact that the product formed is in general a mixture of the two (.alpha. and .beta.) anomeric forms.
Moreover, it is necessary to point out that the intermediary products have to be purified, either by crystallization, or by chromatography, which contributes significantly to the total duration of the synthesis. Finally, it should be noted that the yields may be very weak for certain steps because of steric hindrance, which is specifically the case at the level of the branches: 2 sugars substituting a single monosaccharide.
All in all, this global synthesis is very costly and takes a long time.
In the case of the formation of a glycosylamine followed by acylation, this way depends on a reaction described at the end of the last century: an oside possessing a reducing sugar, incubated in the presence of an elevated concentration of ammoniac, of an ammonium salt or an aromatic amine, is transformed in a reversible manner into glycosylamine.
For example, the lactose gives a lactosylamine, with ammoniac: EQU Gal.beta.4Glc+NH.sub.3.fwdarw.Gal.beta.4Glc.beta.-NH.sub.2 +H.sub.2 O
or with the aniline: EQU Gal.beta.4Glc+NH.sub.2 --C.sub.6 H.sub.5.fwdarw.Gal.beta.4Glc.beta.-NH--C.sub.6 H.sub.5 +H.sub.2 O
This reaction is however reversible, which is to say the isolated product, dissolved in a buffer, becomes hydrolyzed leading to give back the original products.
The glycosylamine may however be stabilized by acylation, for example by selective N-acetylation: EQU Gal.beta.4Glc.beta.-NH.sub.2 +(CH.sub.3 --CO).sub.2 --O.fwdarw.Gal.beta.4Glc.beta.-NH--CO--CH.sub.3 +CH.sub.3 --CO.sub.2 H
On these bases, it has recently been proposed to substitute the amine of oligosylamines by an organic compound possessing a finctional reactive group. Manger et al., 1992, Biochemistry 31, 10724 and 31, 10733.
This route comprises the following steps:
1) incubation of the oside possessing a terminal reducing sugar in the presence of a highly concentrated ammonium salt.
For example, EQU Gal.beta.4Glc+NH.sub.4.revreaction.Gal.beta.4Glc.beta.-NH.sub.3.sup.+ +H.sub.2 O EQU C.sub.12 H.sub.22 O.sub.11 +NH.sub.4.revreaction.C.sub.12 H.sub.24 O.sub.10 N+H.sub.2 O
2) Purification of the glycosylamine by chromatography on a column to eliminate the ammonium salt excess.
3) Substitution of the amine of the glycosylamine by reaction with an activated derivative of monochloroacetic acid, in alkaline medium.
For example, EQU Gal.beta.4Glc.beta.NH.sub.2 +(Cl--CH.sub.2 --CO).sub.2 --O.fwdarw.Gal.beta.4Glc--NH--CO--CH.sub.2 --Cl
4) Transformation of the chloroacetyl residues into glycyl residue.
The chloroacetylglycosylamide is incubated in the presence of a highly concentrated ammonium salt, which allows the introduction of an amine function. For example: EQU Gal.beta.4Glc.beta.-NH--CO--CH.sub.2 --Cl+NH.sub.4.sup.+.fwdarw.Gal.beta.4Glc.beta.-NH--CO--CH.sub.2 --NH.sub.3.sup.+, Cl.sup.- H.sup.+
5) Purification of glycyl-glycosylamide by chromatography on column to eliminate the ammonium salts.
6) Condensation of the glycyl-glycosylamide and of a compound able to selectively react with an amine group of the glycyl residue.
For example: EQU Gal.beta.4Glc.beta.-NH--CO--CH.sub.2 --NH.sub.2 +R--CO-G.fwdarw.Gal.beta.4Glc.beta.-NH--CO--CH.sub.2 --NH--CO--R+HG
in which G is an activator of the carboxylic function.
This route in 6 steps implies two steps of intermediary purification and the use of a toxic product: an activated derivative of chloroacetic acid.
The yield of each step is variable and ranges between 50 and 95%; the global yield is less than approximately 60%.
Another pathway has also been suggested, but it requires the transformation of the reducing sugar into polyol; this route was proposed in 1974 by Gray (Arch. Biochem. Biophys., 163, 426-428).
The oligoside is condensed with a compound comprising one (or many) amine function(s) in the presence of sodium cyanoborohydride in alkaline medium; the imine formed between the reducing sugar and the amine is reduced by the sodium cyanoborohydride into a secondary amine.
For example: EQU Gal.beta.4Glc+NH.sub.2 --R.fwdarw. EQU Gal.beta.4-O--CH(CHOH--CH.sub.2 OH)--CHOH--CHOH--CH.dbd.N--R EQU +NaBH.sub.3 CN.fwdarw.Gal.beta.4-O--CH(CHOH--CH.sub.2 OH)--CHOH--CHOH--CH.sub.2 --NH--R
The yield of this reaction varies according to the size of the partners and is usually between 5 and 70%.
There is a destruction of the reducing sugar, which is not desirable.
French patent number 2 227 823 concerns the N-osides of L pyrrolidone-2-carboxylic-5 acid, derivatives of said acid and their procedure of preparation, consisting of condensing the L pyrrolidone-2-carboxylic-5 acid and/or the L-glutamic acid and/or a salt of these acids with an ose or an oside, with reducing or non-reducing properties.
Following the examples, the reducing sugars are all monosaccharides (ketoses or aldoses), while the non-reducing sugars are, for example, saccharose; taking into consideration the reaction conditions, the saccharose becomes hydrolyzed into glucose and fructose.
It should also be noted that, in the procedure of the said French patent number 2 227 823, condensation takes place preferably in an aqueous medium, at a temperature between 50.degree. C. and 150.degree. C., preferably at approximately 105.degree. C.
These operating conditions require working with very concentrated solutions of sugar and acid which are inapplicable in the preparation of oligosides; the solubility of oligosides decreases rapidly while the number of sugars increases.
Furthermore, the condensation reaction between the sugar and the acid being a thermic dehydration, it is inapplicable to the preparation of oligosides because of the fragility of the oside linkages, which are easily hydrolyzed in hot conditions (see for example the case of saccharose).
Thermic condensation presents, moreover, the drawback of giving colored products, which are the evidence of degradation reactions and require a further step of purification.
The procedure thus does not allow obtaining anything but derivatives of monosaccharides and is not adapted to the production of derivatives of oligosides.